Crystallographic studies on the binding of coenzyme analogs to D-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor

In contrast with the coezyme, two coenzyme analogs, ADP-ribose and SNAD, bind non-cooperatively to D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Palinurus versicolor (PV) GAPDH complexed with ADP-ribose and SNAD has been crystallized by the method of sitting-drop vapor diffusion. X-ray diffraction data analysis reveals that both crystals belong to the same space group (C2), and have similar cell dimensions: a =152.80 A, b =100.35 A, c =128.31 A, β=110.28° and a =153.41 A, b =100.51 A, c =128.44 A, β =110.48°, respectively. It is estimated that the asymmetric unit in each crystal contains 4 subunits. This is a novel crystal form which is quite different from that previously reported for holo- and apo-GAPDH from the same spurce. The result suggests that the binding of the two coenzyme analogs to GAPDH may lead to some significant conformational changes, which are different from those induced by the coenzyme binding. The self-rotation function indicates that the tetramer of these two GAPDH

Structure of D-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor in a tetragonal crystal form

D-glyceraldehyde-3-phosphate dehydrogenase (holo-GAPDH) from Palinurus versicolor was crystallized in a novel crystal form by the method of sitting-drop vapor diffusion. The crystals have space group P4212, cell parameters a=15.49 nm, c=8.03 nm and two subunits per asymmetric unit. The crystal structure at 0.34 nm was determined by the molecular replacement method. The final model has crystallographic Rfree and R factors of 0.274 and 0.262, and r.m.s. deviations of 0.002 nm for bond lengths and 2.33?for bond angles. The two subunits in asymmetric unit are similar to each other not only in the three-dimensional structure, but also in average temperature factors. This result demonstrates that the obvious difference in average temperature factors for the different subunits in C2 crystal form reported previously may be attributed to the different crystallographic environments of the subunits. This further supports that holo-GAPDH has a good 222 molecular symmetry.

Separation and Purification of GST-glycerol-3-phosphate Dehydrogenase

In order to investigate the expression of glycerol-3 -phosphate dehydrogenase by GCY1 gene in recombinant Saccharomyces cerevisiae, induction culture of the S. cerevisiaestrain was performed with SD-URA 2% galactose, 3 × YP ＋ 6% glucose, SC-URA 2% galactose, and SC-URA 2% galactose ＋ 5% NaCI glyeerol-3-phosphate dehydregenase, the cultured S. cerevisiaewas comminuted followed by full-automatic high-speed purification, and SDS-PAGE gel electrophoresis was performed for molecular weight of the GST fusion protein. The results showed that after shaking culture of the S. cerevisiae containing GCY1 at 25 ℃, the OD values of its 3 × YP ＋ 6% glucose culture and SC-URA 2% galaetose ＋ 5% NaC1 culture were 8.75 and 7.35, respectively. It was shown by purification with a Profinia low-pressure liquid chromatograph that only the S. cerevisiae cultured in SC-URA 2% galactose ＋ 5% NaC1 medium expressed glycerel-3-phosphate de- hydrogenase, the molecular weight of which was detected as 65 ku by SDS-PAGE gel electrophoresis.

Role of Cathepsin G in the Degradation of Glyceraldehyde-3-Phosphate Dehydrogenase Triggered by 4-Hydroxy-2-Nonenal in U937 Cells

Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, we examined whether GAPDH in U937 cells treated with HNE in culture is degraded similarly to that incubated with HNE and U937 cell extract. Treatment with HNE for 10 min in culture decreased GAPDH activity in a concentration dependent manner, but did not affect GAPDH degradation. The proteasome activities were not affected by HNE, but culturing with HNE decreased cathepsin G activity and protein level in a concentration dependent manner. These results suggest that HNE-induced oxidative stress leads to decreased cathepsin G activity and results in the loss of GAPDH degradation. Taken together, our findings indicate that cathepsin G has an important role in the degradation of oxidatively modified GAPDH in U937 cells.

Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from saline strain Idiomarina loihiensis

Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterization of glyceralehyde-3-phosphate dehydrogenase (GAPDH) fromI. loihiensis saline’s bacteria on the basis of the apparent native and subunit molecular weights, physico-chemical and kinetic characterizations. The purification method consisted of two steps, ammonium sulfate fractionation followed by one chromatographic step, namely dye-affinity on Blue Sepharose CL-6B. Polyclonal antibodies against the purified enzyme were used to recognize theI. loihiensis GAPDH by Western blotting. The optimum pH of the purified enzyme was 8.5. Studies on the effect of temperatures revealed an enzyme increasing activity of about 45?C. The molecular weight of the purified enzyme was 36 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yield a molecular weight of 147 kDa. The Michaelis constants for NAD+ and D-glyceraldehyde-3-phosphate estimated was 19 μM and 3.1 μM, respectively. The maximal velocity of the purified enzyme was estimated to be 2.06 U/mg, approximately 6-fold increase in specific activity and a final yield of approximately 32.5%. The physicochemical properties of this GAPDH, being characterized, could be used in further studies.

Sperm glyceraldehyde 3-phosphate dehydrogenase gene expression in asthenozoospermic spermatozoa

It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have investigated the chemical and kinetic properties of the enzyme glyceraldehyde 3-phosphate dehydrogenase to identify any changes in the regulation of glycolysis and sperm motility. In contrast, there are few studies analyzing the genetic basis of hypokinesis. For this reason, we investigated the glyceraldehyde 3-phosphate dehydrogenase gene in human sperm to evaluate whether asthenozoospermia was correlated with any changes in its expression. Semen examination and glyceraldehyde 3-phosphate dehydrogenase gene expression studies were carried out on 116 semen samples divided into two groups - Group A consisted of 58 normokinetic samples and Group B of 58 hypokinetic samples. Total RNA was extracted from spermatozoa, and real-time PCR quantification of mRNA was carried out using specific primers and probes. The expression profiles for the Groups A and B were very similar. The mean delta Ct was as follows - Group A, 5.79 ＋ 1.04; Group B, 5.47 ＋ 1.27. Our study shows that in human sperm, there is no difference in glyceraldehyde 3-phosphate dehydrogenase gene expression between samples with impaired motility and samples with normal kinetics. We believe that this study could help in the understanding of the molecular mechanisms of sperm kinetics, suggesting that hypomotility may be due to a possible posttranscriptional impairment of the control mechanism, such as mRNA splicing, or to posttranslational changes.

Effect of mango seed kernel extract on the adipogenesis in 3T3-L1 adipocytes and in rats fed a high fat diet

Mangoes (Mangifera indica L.) are one of the most important tropical foods. The seed is one of the main by-products of mango processing. Therefore, it is important to find an economically viable use for this waste (e.g., as a food additive or supplement with high nutraceutical value). We investigated the anti-obesity effects of mango seed kernel extract with hot water (MSKE-W) in 3T3-L1 adipocytes and in a high fat diet (HFD)-induced obesity rat model. MSKE-W caused a significant decrease in the activity of glycerol 2-phosphate dehydrogenase in 3T3-L1 adipocytes without eliciting cell cytotoxicity and inhibited cellular lipid accumulation through down-regulation of transcription factors such as PPARγ and C/EBPα. In the animal model, rats fed an HFD containing 1% MSKE-W gained less weight than rats fed an HFD alone. The visceral fat mass in rats fed an HFD containing 1% MSKE-W tended to be lower than that in rats fed an HFD alone. Furthermore, histological examination of rat livers from an HFD showed steatohepatitis. However, rats on an HFD containning 1% MSKE-W showed no histopathological changes in liver tissue. Our results indicate that MSKE-W influences anti-obesity effects, both in vitro and in vivo, and suggest that MSKE-W provides a novel preventive potential against obesity.

The role of AtGPDHc2 in regulating cellular redox homeostasis of Arabidopsis under salt stress

Plants glycerol-3-phosphate dehydrogenase(GPDH)catalyzes the formation of glycerol-3-phosphate,and plays an essential role in glycerolipid metabolism and stress responses.In the present study,the knock-out mutants of cytosolic GPDH(AtGPDHc2)and wild-type Arabidopsis plants were treated with 0,50,100,and 150 mmol L–1 NaCl to reveal the effects of AtGPDHc2 deficiency on salinity stress responses.The fluctuation in redox status,reactive oxygen species(ROS)and antioxidant enzymes as well as the transcripts of genes involved in the relevant processes were measured.In the presence of 100 and 150 mmol L–1 NaCl treatments,AtGPDHc2-deficient plants exhibited a pronounced reduction in germination rate,fresh weight,root length,and overall biomass.Furthermore,loss of AtGPDHc2 resulted in a significant perturbation in cellular redox state(NADH/NAD+and AsA/DHA)and consequent elevation of ROS and thiobarbituric acid-reactive substances(TBARS)content.The elevated ROS level triggered substantial increases in ROS-scavenging enzymes activities,and the up-regulated transcripts of the genes(CSD1,sAPX and PER33)encoding the antioxidant enzymes were also observed.In addition,the transcript levels of COX15,AOX1A and GLDH in gpdhc2 mutants decreased in comparison to wild-type plants,which demonstrated that the deficiency of AtGPDHc2 might also has impact on mitochondrial respiration under salt stress.Together,this work provides some new evidences on illustrating the roles of AtGPDHc2 playing in response to salinity stress by regulating cellular redox homeostasis,ROS metabolism and mitochondrial respiration.

The Effect of Ultra Low Concentrations of Some Biologically Active Substances on the Aerobic Respiration

For today it is known, that primary and secondary disorders of the aerobic respiration, which are based on mitochondrial deficiency, lead to a wide spectrum of clinical manifestations and diseases. Therefore, the question about effective correction of various types of energy exchange disorders remains topical. Thus, the aim of our work was the study effect of the complex of biologically active substances (BAS) in ultra low concentrations on the activity of key enzymes of aerobic energy metabolism succinate dehydrogenase (EC 1.3.99.1) (SQR) and mitochondrial glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) (GPD2). The human lymphocytes assays were tested in vitro (22 donors). In negative control lymphocytes, cell culture normal saline solution was added. Normal saline solution with NaN3 was added in positive control lymphocytes cell culture. Experimental cell culture contained NaN3 and BAS. Our investigations had been revealed increase SQR activity in the experimental cell culture as compared with positive control culture throughout the time of experiment (measurements were carried out at 4, 24, 48, 72 h of incubation). The SQR activity of experimental cell culture and negative control lymphocytes cell culture was equal up to 24 h of experiment. It showed neutralization of NaN3 inhibitory effect (during 24 h) due to BAS influence. Activity of base glycerophosphate shunt ferment GPD2 of experimental lymphocyte cell culture was not different from GPD2 index in the negative control, but was lower than GPD2 activity in the positive control. It also indicated neutralization NaN3 inhibitory effect due to BAS influence. So we had found that extremely low concentrations of selected BAS with their complex impact on human lymphocytes in vitro could effectively neutralize the inhibitory effect of NaN3 on processes of aerobic energy metabolism link.